The goal of this Phase II project is to optimize and validate a microfluidic chip-based process that automates and shortens the labeling, washing and collecting of human blood leukocytes (WBCs) for flow cytometric analysis of clinically important cell-surface and intracellular markers. Multi-parameter flow cytometry is an increasingly powerful and widely used technology in research and in clinical diagnostic testing for cancer and many other diseases. There is a critical need for new methods to improve the efficiency of sample preparation, which is laborious and variable and typically requires many cycles of washing collecting cells by centrifugation. GPB LLC is developing a novel microfluidic Deterministic Lateral Displacement (DLD) chip processing technology that can harvest cells from a flow of fluid on the basis of size. A mixture of fluid and particles flows through an array f microposts that are tilted at a small angle from the direction of the fluid flow. Cells within the target size range are deflected off the microposts in a process that is rapid yet gentle. The novel disposable DLD chips will allow automation and potentially combination of the processes of labeling, fixing and washing cells from very small samples of whole blood. The Phase I STTR project, a collaboration among GPB, Princeton University and the University of Maryland (UM), successfully demonstrated proof of principle by showing that prototype DLD chips can harvest monoclonal antibody (Mab)-labeled WBCs from human whole blood with high cell recovery and viability while removing the large excess of red blood cells (RBCs) and unbound Mab, and without skewing the distribution of WBC subsets. The objective of this Phase II project is to optimize a suite of DLD chips and processes as prototype commercial products. Aim 1 is to design and optimize DLD chip-based systems for washing and concentrating human blood WBCs. Aim 2 is to design and validate DLD chip-based systems for surface labeling, on-chip fixation-permeabilization, or intracellular labeling of WBCs, combined with on-chip washing. Aim 3 is to develop a DLD chip-based system to fix, permeabilize, intracellularly label, wash, and concentrate WBCs on a single chip. Aim 4 is to produce prototype application-specific chips and an automated platform product to control WBC processing via a user interface. Cell labeling will be tested with panels of Mab reagents and cocktails that are commonly used for cell surface labeling and for intracellular labeling in clinical diagnostic, prognostic and research testing. Outcomes will be measured by flow cytometry in several laboratories at UM, GBP and several collaborating large diagnostic companies. Some of these companies have expressed interest in helping to commercialize chip and processing system products.